Glycosylation is one of the most prominent protein modifications and many if not most secretory and membrane-bound proteins produced by mammalian cells contain covalently linked glycans (Varki, A. et al. Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, 2009). In the assembly of complex organisms such oligosaccharide portions serve a variety of structural and functional roles for the folding, subcellular localization, turnover, activity and interactions of secreted and cell surface proteins.
Secreted glycoproteins include e.g. cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, antigen recognition molecules, immunoregulatory molecules, structural glycoproteins, and other bioactive molecules. Those proteins are important in many recognition events, such as cell-to-cell signaling, immune responses, apoptosis, host-pathogen interactions and the pathogenesis of many diseases. Thereby, the specificity of such glycoproteins for certain target receptors is essential in regulating cell-to-cell communication. Thus the identification and characterization of ligand binding interactions of secreted glycoproteins with their targets is essential for a molecular understanding of biological information transfer.
In analogy, the engagement of cell surface glycoprotein receptors (CSRs) by ligands, such as proteins, peptides, hormones, chemical molecules, pharmaceutical drugs or toxins enables the transfer of information from the cellular microenvironment into the cell. Despite the fact that this cell surface information gateway is critical for cellular responses, the receptors for many functional ligands remain unknown. This is mainly due to technological limitations in the identification of hydrophobic membrane receptor proteins and due to transient, low affinity interactions of ligands with their corresponding CSRs. Therefore, many signaling proteins and molecules remain orphan ligands without a known primary molecular target—invaluable information currently missing for a detailed molecular understanding of the respective mechanisms of signal transduction, drug action, off-target effects or disease-associated signaling networks.
A promising approach to the identification of transient ligand-receptor interactions in biological systems is the chemical crosslinking of interacting molecules followed by mass spectrometric identification of the interaction partners. Currently known and commercially available crosslinkers have typically been designed for their use in mapping protein interfaces with isolated proteins in solution. For example, homobifunctional or heterobifunctional crosslinkers (including cleavable or isotope-encoded derivatives) have been used for the chemical crosslinking of proteins followed by enzymatic digestion and mass spectrometric identification of the crosslinked peptides for mapping three-dimensional structures of proteins and protein complexes (JMS (2003) vol. 38 (12) pp. 1225-37). However, the crosslinked peptide species obtained with such molecules are typically of very low relative abundance and the bioinformatic analysis of the mass spectra produced by crosslinked peptides remains a daunting task. This hampers the identification of crosslinking sites in complex biological samples, in particular for the detection of typically transient interactions of ligands with their corresponding receptors.
In order to specifically enrich crosslinked peptides out of complex samples, trifunctional crosslinkers have been disclosed having a combination of two reactive sites (typically amine-reactive, sulfhydryl-reactive or photoreactive) to capture interacting proteins, and an affinity group (typically biotin) for the subsequent enrichment of captured peptides (J Am Soc Mass Spectrom (2005) vol. 16 (12) pp. 1921-31). While these crosslinkers have been used successfully for the mapping of topological structures of isolated proteins and protein complexes, their chemical nature renders them unsuitable for the detection of transient protein-protein interactions in complex samples derived from live cells.
This highlights the need for suitable reagents that are able to aid specifically in the probing, identification, and characterization of ligand interactions with target glycoprotein receptors in their biological microenvironment, such as in biological fluids or associated with the plasma membrane of living cells. To applicant's best knowledge, neither of the known crosslinkers today is able to fulfill the structural requirements for enabling the covalent stabilization and subsequent mass-spectrometric identification of specific interactions between a known ligand and unknown glycoprotein receptor binding partners in a complex and natural environment, such as the surface of a living cell.
Applicants have now found that a novel class of trifunctional crosslinking reagents, hereinafter also called crosslinkers of the invention, are able to overcome the problems inherent to the ligand-based identification of target receptors of the prior art. The crosslinkers of the invention can be used for the unbiased detection and characterization of ligand-receptor interactions between a ligand and a target glycoprotein receptor with high sensitivity and specificity on live cells or in biological fluids applying near-physiological conditions. This method can thus be applied to identify unknown target receptors for orphan ligands such as proteins, peptides, lipids, engineered affinity binders, chemical molecules, drugs, viruses or bacteria. Thus, the new crosslinking reagents provide a technological basis for the understanding of the human surfaceome and secretome as a complex information gateway and a means to identify target glycoprotein receptors for orphan ligands of almost every description within their native microenvironment.